The zinc finger antiviral protein (ZAP) is an interferon- and IRF3-induced host protein that inhibits replication of members of several virus families. Although the existence of two isoforms (ZAPS and ZAPL) has been appreciated for some time, new preliminary evidence demonstrates protein expression of at least four isoforms. This project will determine the antiviral spectrum of the four individual isoforms of ZAP: ZAPS, ZAPL and the newly identified ZAPM and ZAPXL against viruses known to be sensitive to ZAP's effect, including the alphaviruses Sindbis virus (SINV), chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV), human immunodeficiency virus type 1 (HIV-1), Ebola virus (EBOV) and hepatitis B virus (HBV). Moreover, the the antiviral activity of the individual isoforms of this important component of the host cell's innate immune response will be characterized for viruses not previously tested for ZAP sensitivity. The effects of the isoforms on translation (usin the SINV genome), miRNA-mediated translational arrest and interferon induction will be ascertained. The studies will determine if the isoforms have unique biological activities and whether these activities require cooperation between isoforms. Finally, the work will determine the contributions of ZAP ADP-ribosylation to ZAP function, and the role of the alphavirus nsP3 macrodomain in ADP-ribose removal and ZAP antagonism. Together the results obtained from these studies will have far reaching impact, affecting areas as diverse as translation control, innate immunity, numerous virus fields, macrodomain function and the role in protein function of ADP- ribosylation. The studies will clarify our understanding of ZAP function, in particular defining whether the different isoforms have unique biological activities. Knowledge gained can be leveraged for the development of safe and effective vaccines as well as novel antiviral approaches including macrodomain antagonists and ZAP mimics.